Preparation of sustained release Vitamin K2 microspheres and evaluation of it’s application for bone tissue engineering

• Main Authors: Min-De Chung, 鍾旻德
• Other Authors: Chun-Mei Lu
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/71137996350742176947

碩士 === 國立勤益科技大學 === 化工與材料工程系 === 100 === In this work, we prepared microspheres containing vitamin K2 (VK2) and poly(lactic–co-glycolic acid) (PLGA) biodegradable polymer with the oil in water (O/W) emulsion nonaqueous phase separation method. First, 0%, 0.01%, 0.1%, and 1.0% (w/v) vitamin K2 were loaded into the PLGA microspheres, signature as VK2MS, and calculated the yield of VK2MS. Then VK2MS were observed by a scanning electron microscope (SEM) to observe its morphology. The laser scattering particle size distribution analyzer (LS), UV/Visible spectrometer (UV/Vis) and Attenuated Total Reflectance Fourier transform infrared spectrometry (ATR-FTIR) were used to determine the effect of VK2 concentration on particle size distribution and encapsulation efficiency. VK2MS were determined with drug release assay for 70 days in vitro. Finally, the cell growth rate and cell number were estimated for 14 days with MG-63 cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and detection cell of bone differentiation situation by the assay of alkaline phosphatase (ALP) activity. Hematoxylin &; Eosin (H&;E) and Alizarin red S staining were utilized to evaluate the effect of VK2MS on MG-63 cells. The yield of VK2MS would be able to increase with VK2 content, 1.0% VK2MS had the heights value of 80.8 ± 6.9%. All microspheres were spherical with a smooth surface, and particle size distribution was in the range of 2~10 μm, their scale might expanded with VK2 content. We confirmed VK2 loaded into the PLGA microspheres, and PVA has been cleaned from ATR-FTIR. The encapsulation efficiency of VK2MS were above 67.6 ± 7.1%, 0.1% VK2MS had the highest value of 92.8 ± 5.2%. In drug release assay, the 0.01% VK2MS showed that the VK2 was almost complete release for 35 days, 0.1% and 1.0% VK2MS had released of 36.6 ± 1.4% and 7.0 ± 0.4% in 70 days respectively. It seems drug release rate was increased by decreasing VK2 loaded into the microspheres. The cumulative percent released showed VK2MS followed the zero-order manner. VK2 was a hydrophobic drug, whose could prevent degradation of PLGA. 0.01% VK2MS had the fastest hydrolysis rate caused by the smallest particle size of 2.9 ± 0.6 μm, speed up the drug release rate. The degradation of VK2MS for 42 days, 0.01% VK2MS had the lowest percentage mass remaining of 33.0 ± 2.2%; whereas 1.0% VK2MS had the highest value of 69.3 ± 1.2% signs could be seen. In vitro of cell proliferation assay, VK2 shown a cytostatic effect, and the inhibitive effect increased with the VK2 concentration, and 1.0 mg/ml VK2 by which MG-63 cells for 7 days was most inhibited in cell growth rate of 17.4 ± 0.4%; 0.002 mg/ml VK2 could increase the ALP activity, which treatment of osteosarcoma cells for 3 days had the heights value of 13.7 ± 0.1 U/L-30 min. On the other hand, VK2MS also inhibited in cell proliferation, 1.0% VK2MS by which MG-63 cells for 7 days was most inhibited in cell growth rate of 44.5 ± 0.3%, but its inhibitory effect was more less than that of VK2; VK2MS could promote cell differentiation, enhanced the ALP activity of single cell, and 0.1% VK2MS treatment of osteosarcoma cells for 3 days had the heights value of 11.2 ± 0.0 U/L-30 min. This situation was demonstrated by histochemical staining method for H&;E and Alizarin red S. The VK2MS enhanced bone regeneration was made successfully, and it is respected to apply for bone regeneration in clinic in the future.